scholarly journals Dodging COVID-19 Infection: Low Expression and Localization of ACE2 and TMPRSS2 in Human Umbilical Cord-Derived Mesenchymal Stem Cells

2021 ◽  
Author(s):  
Jonathan J Hernandez ◽  
Doyle E Beaty ◽  
Logan L Fruhwirth ◽  
Ana P Lopes Chaves ◽  
Neil H Riordan
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Western Blot ◽  
Viral Infection ◽  
Umbilical Cord ◽  
Quantitative Polymerase Chain Reaction ◽  
Human Umbilical Cord ◽  
Human Lung Tissue ◽  
Novel Coronavirus ◽  
Low Expression

Abstract BackgroundMesenchymal stem cells derived from human umbilical cord (hUC-MSCs) have immunomodulatory properties that are of interest to treat novel coronavirus disease 2019 (COVID-19). Leng et al. recently reported that hUC-MSCs derived from one donor negatively expressed Angiotensin-Converting Enzyme 2 (ACE2), a key protein for viral infection along with Transmembrane Serine Protease 2 (TMPRSS2). MethodsExpression of ACE2 and TMPRSS2 was analyzed in 24 lots of hUC-MSCs derived fromWharton's jelly via quantitative polymerase chain reaction (qPCR), Western Blot, immunofluorescence and flow cytometry using 24 different donors. ResultshUC-MSCs had significantly lower ACE2 (p=0.002) and TMPRSS2 (p=0.008) expression compared with human lung tissue homogenates in Western blot analyses. Little to no expression of ACE2 was observed in hUC-MSC by qPCR, and they were not observable with immunofluorescence in hUC-MSCs cell membranes. A negative ACE2 and TMPRSS2 population percentage of 95.3% ±15.55 was obtained for hUC-MSCs via flow cytometry, with only 4.6% ACE2 and 29.5% TMPRSS2 observable positive populations. ConclusionsWe have demonstrated negative expression of ACE2 and low expression of TMPRSS2 in 24 lots of hUC-MSCs. This has crucial implications for the design of future therapeutic options for COVID-19, since hUC-MSCs would have the ability to “dodge” viral infection to exert their immunomodulatory effects.

scholarly journals Dodging COVID-19 infection: low expression and localization of ACE2 and TMPRSS2 in multiple donor-derived lines of human umbilical cord-derived mesenchymal stem cells

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jonathan J. Hernandez ◽  
Doyle E. Beaty ◽  
Logan L. Fruhwirth ◽  
Ana P. Lopes Chaves ◽  
Neil H. Riordan
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Western Blot ◽  
Viral Infection ◽  
Umbilical Cord ◽  
Quantitative Polymerase Chain Reaction ◽  
Human Umbilical Cord ◽  
Human Lung Tissue ◽  
Low Expression

Abstract Background Mesenchymal stem cells derived from human umbilical cord (hUC-MSCs) have immunomodulatory properties that are of interest to treat novel coronavirus disease 2019 (COVID-19). Leng et al. recently reported that hUC-MSCs derived from one donor negatively expressed Angiotensin-Converting Enzyme 2 (ACE2), a key protein for viral infection along with Transmembrane Serine Protease 2 (TMPRSS2). The purpose of this study was to quantify the expression of ACE2 and TMPRSS2 in hUC-MSCs lots derived from multiple donors using molecular-based techniques in order to demonstrate their inability to be a host to SARS-CoV-2. Methods Expression of ACE2 and TMPRSS2 was analyzed in 24 lots of hUC-MSCs derived from Wharton's jelly via quantitative polymerase chain reaction (qPCR), Western Blot, immunofluorescence and flow cytometry using 24 different donors. Results hUC-MSCs had significantly lower ACE2 (p = 0.002) and TMPRSS2 (p = 0.008) expression compared with human lung tissue homogenates in Western blot analyses. Little to no expression of ACE2 was observed in hUC-MSC by qPCR, and they were not observable with immunofluorescence in hUC-MSCs cell membranes. A negative ACE2 and TMPRSS2 population percentage of 95.3% ± 15.55 was obtained for hUC-MSCs via flow cytometry, with only 4.6% ACE2 and 29.5% TMPRSS2 observable positive populations. Conclusions We have demonstrated negative expression of ACE2 and low expression of TMPRSS2 in 24 lots of hUC-MSCs. This has crucial implications for the design of future therapeutic options for COVID-19, since hUC-MSCs would have the ability to “dodge” viral infection to exert their immunomodulatory effects.

scholarly journals Dodging COVID-19 infection: Low expression and localization of Angiotensin-Converting Enzyme 2 (ACE2) and Transmembrane Serine Protease 2 (TMPRSS2) in Mesenchymal Stem Cells derived from human umbilical cord (hUC-MSCs). 

2020 ◽  
Author(s):  
Jonathan J Hernandez ◽  
Doyle E Beaty ◽  
Logan L Fruhwirth ◽  
J M Sloan ◽  
Ana P Lopez Chaves ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Angiotensin Converting Enzyme ◽  
Western Blot ◽  
Human Umbilical Cord ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Transmembrane Serine Protease ◽  
Low Expression

Abstract Mesenchymal stem cells derived from human umbilical cord (hUC-MSCs) have immunomodulatory properties that are of interest to treat novel coronavirus disease 2019 (COVID-19). Leng et al. recently reported that hUC-MSCs derived from one donor negatively expressed Angiotensin-Converting Enzyme 2 (ACE2), a key protein for viral infection along with Transmembrane Serine Protease 2 (TMPRSS2). In this report, the expression of ACE2 and TMPRSS2 was analyzed in 24 lots of hUC-MSCs derived from 24 different donors via quantitative polymerase chain reaction (qPCR), Western Blot, immunofluorescence and flow cytometry. hUC-MSCs had significantly lower ACE2 (p=0.002) and TMPRSS2 (p=0.008) expression compared with human lung tissue homogenates in Western blot analyses. Little to no expression of ACE2 or TMPRSS2 was observed in hUC-MSC by qPCR, and they were not observable with immunofluorescence in hUC-MSCs cell membranes. A double negative ACE2 and TMPRSS2 population percentage of 94.30% ±15.55 was obtained for hUC-MSCs via flow cytometry, with only 0.011% ACE2 and 10.91% TMPRSS2 observable positive populations. We have demonstrated negative expression of ACE2 and low expression of TMPRSS2 in 24 lots of hUC-MSCs. This has crucial implications for the design of future therapeutic options for COVID-19, since hUC-MSCs would have the ability to “dodge” viral infection to exert their immunomodulatory effects.

scholarly journals Human Umbilical Cord Mesenchymal Stem Cells Inhibit the Function of Allogeneic Activated Vγ9Vδ2 T Lymphocytes In Vitro

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Xiaohuan Liu ◽  
Ting Feng ◽  
Tianxiang Gong ◽  
Chongyang Shen ◽  
Tingting Zhu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
T Cells ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
Umbilical Cord ◽  
Interleukin 2 ◽  
Cell Contact ◽  
Human Umbilical Cord ◽  
Fas L

Background. Human umbilical cord mesenchymal stem cells (UC-MSCs) can regulate the function of immune cells. However, whether and how UC-MSCs can modulate the function of Vγ9Vδ2 T cells has not been fully understood. Methods. The PBMCs or Vγ9Vδ2 T cells were activated and expanded with pamidronate (PAM) and interleukin-2 (IL-2) with or without the presence UC-MSCs. The effects of UC-MSCs on the proliferation, cytokine expression, and cytotoxicity of Vγ9Vδ2 T cells were determined by flow cytometry. The effects of UC-MSCs on Fas-L, TRAIL-expressing Vγ9Vδ2 T cells, and Vγ9Vδ2 T cell apoptosis were determined by flow cytometry. Results. UC-MSCs inhibited Vγ9Vδ2 T cell proliferation in a dose-dependent but cell-contact independent manner. Coculture with UC-MSCs reduced the frequency of IFNγ+ but increased granzyme B+ Vγ9Vδ2 T cells. UC-MSCs inhibited the cytotoxicity of Vγ9Vδ2 T cells against influenza virus H1N1 infected A549 cells and also reduced the frequency of Fas-L+, TRAIL+ Vγ9Vδ2 T cells but failed to modulate the apoptosis of Vγ9Vδ2 T cells. Conclusions. These results indicated that UC-MSCs efficiently suppressed the proliferation and cytotoxicity of Vγ9Vδ2 T cells and modulated their cytokine production. Fas-L and TRAIL were involved in the regulation. Cell contact and apoptosis of Vγ9Vδ2 T cells were not necessary for the inhibition.

scholarly journals Simulated Microgravity Reduces Proliferation and Reorganizes the Cytoskeleton of Human Umbilical Cord Mesenchymal Stem Cells

2020 ◽  
pp. 897-906
Author(s):  
H CHI ◽  
H SON ◽  
D CHUNG ◽  
L HUAN ◽  
T DIEM ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Western Blot ◽  
Umbilical Cord ◽  
Simulated Microgravity ◽  
Cellular Proliferation ◽  
Cytoskeletal Proteins ◽  
Control Group ◽  
Human Umbilical Cord

The cytoskeleton plays a key role in cellular proliferation, cell-shape maintenance and internal cellular organization. Cells are highly sensitive to changes in microgravity, which can induce alterations in the distribution of the cytoskeletal and cell proliferation. This study aimed to assess the effects of simulated microgravity (SMG) on the proliferation and expression of major cell cycle-related regulators and cytoskeletal proteins in human umbilical cord mesenchymal stem cells (hucMSCs). A WST-1 assay showed that the proliferation of SMG-exposed hucMSCs was lower than a control group. Furthermore, flow cytometry analysis demonstrated that the percentage of SMG-exposed hucMSCs in the G0/G1 phase was higher than the control group. A western blot analysis revealed there was a downregulation of cyclin A1 and A2 expression in SMG-exposed hucMSCs as well. The expression of cyclin-dependent kinase 4 (cdk4) and 6 (cdk6) were also observed to be reduced in the SMG-exposed hucMSCs. The total nuclear intensity of SMG-exposed hucMSCs was also lower than the control group. However, there were no differences in the nuclear area or nuclear-shape value of hucMSCs from the SMG and control groups. A western blot and quantitative RT-PCR analysis showed that SMG-exposed hucMSCs experienced a downregulation of β-actin and α-tubulin compared to the control group. SMG generated the reorganization of microtubules and microfilaments in hucMSCs. Our study supports the idea that the downregulation of major cell cycle-related proteins and cytoskeletal proteins results in the remodeling of the cytoskeleton and the proliferation of hucMSCs.

scholarly journals DAPT inhibits the chondrogenesis of human umbilical cord mesenchymal stem cells

2015 ◽  
Vol 10 (1) ◽  
Author(s):  
Xiaoke Hu ◽  
Jiawen Zhang ◽  
Xinxin Shao ◽  
Ermei Luo ◽  
Li Yu
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Notch Signaling ◽  
Umbilical Cord ◽  
Quantitative Polymerase Chain Reaction ◽  
Butyl Ester ◽  
Notch Pathway ◽  
Human Umbilical Cord ◽  
Notch 1

AbstractNotch signaling plays a key role in cell proliferation and differentiation, and is important in several biological processes, but its role in the chondrogenesis of human umbilical cord mesenchymal stem cells (UC-MSCs) is still unknown. N-[N-(3,5- difluorophenacetyl-L-alanyl)]-(S)-phenylglycinet-butyl ester (DAPT) is the inhibitor of Notch pathway. The aim of this study is to investgate the influence of DAPT on the chondrogenesis of UC-MSCs. In our study, UC-MSCs were isolated from human umbilical cord and their characteristics were identified. The UC-MSCs were induced to differentiate into chondrocytes in vitro and treated with 5 μM DAPT. Glycosaminoglycan (GAG) and collagen type II (COL-2A1) were analyzed qualitatively and quantitatively. The gene expression of Notch-1, Hes-1, GAG and COL-2A1 were analyzed by quantitative polymerase chain reaction (qPCR). The UC-MSCs separated from human umbilical cord, followed the characteristics of Mesenchymal Stem Cells (MSCs). The gene expression of Notch-1 and Hes-1 decreased after chondrogenic induction but the percentage in G1 period and the content of GAG and COL-2A1 increased. The expression of all tested Notch signaling and proliferation genes declined when 5 μM DAPT was added, also the content of GAG and COL-2A1 also decreased. Our study revealed that Notch signaling exists in UC-MSCs and it may remain the proliferative activity of UC-MSCs. Once the chondrogenesis begins, Notch signaling strength decline evidently. DAPT inhibits the chondrogenesis of UC-MSCs.

scholarly journals The Effects of Indoxyl Sulfate on Human Umbilical Cord-Derived Mesenchymal Stem Cells In Vitro

2016 ◽  
Vol 38 (1) ◽  
pp. 401-414 ◽  
Author(s):  
Wei Wang ◽  
Xueyong Liu ◽  
Wei Wang ◽  
Jinghua Li ◽  
Yuanyuan Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Indoxyl Sulfate ◽  
Proliferative Potential ◽  
Human Umbilical Cord ◽  
Uremic Toxin ◽  
Ki 67

Background/Aims: Indoxyl sulfate, an important protein-bound uremic toxin, can damage stem cells, thus hampering stem cell-based regenerative medicine approaches targeting chronic kidney diseases (CKD). Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are thought to have promising clinical application because of their high proliferative potential and ease of isolation than MSCs from other sources. In the present study, we aimed to determine the harmful effects of indoxyl sulfate on the phenotype and functional potential of hUC-MSCs in vitro. Methods: The toxicity and cell viability was examined by Trypan blue exclusion and MTT assay. The cellular surface markers and the percentage of apoptotic cells by Annexin-V/PI staining were analyzed by flow cytometry. Proliferation was evaluated based on cell number counting and Ki-67 immunostaining. Cell senescence was measured using senescence-associated β-Galactosidase activity. The ability to stimulate the development of CD4+CD25+FoxP3+ regulatory T cells was assessed by incubating hUC-MSCs with peripheral blood mononuclear cells from the healthy volunteers. Results: Our results demonstrated that the immunophenotype of hUC-MSCs was not affected by indoxyl sulfate flow cytometry. However, a significant decrease in cell numbers and fraction of Ki-67 positive proliferating cells, along with a significant increase in cellular senescence were detected in hUC-MSCs after exposure to indoxyl sulfate. Additionally, their ability to stimulate CD4+CD25+FoxP3+ regulatory T cell production was compromised when hUC-MSCs were pretreated with indoxyl sulfate. Conclusion: Taken together, our study clearly demonstrated that the molecular alterations and functional incompetence in hUC-MSCs under the challenge of indoxyl sulfate in vitro.

scholarly journals Hematopoietic Reconstitution of Human Umbilical Cord Mesenchymal Stem Cells after Leukemia Chemotherapy: Effectiveness and Safety

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2171-2171
Author(s):  
Yuxin Tan ◽  
Lu Ding ◽  
Can Can ◽  
Fuling Zhou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Peripheral Blood ◽  
The Body ◽  
Human Umbilical Cord ◽  
Through Flow ◽  
Pdx Models

Abstract Objective: As the incidence of tumors increases, more patients need chemotherapy. Patients receiving chemotherapy also inevitably suffer side effects from treatment. Damage to the immune and hematopoietic system causes the failure of therapies. Mesenchymal stem cells have excellent capabilities in immune regulation and hematopoietic support. This study preliminarily explored the safety and effectiveness and of human umbilical cord mesenchymal stem cells (UCMSC) in myelosuppressive patient-derived tumor xenograft (PDX) models of acute myeloid leukemia (AML). Methods: B-NDG mice were used to establish the PDX models. The mice were randomly divided into three groups: AML group, AML+Ara-C group and AML+Ara-C+MSC group. Mice in the AML+Ara-C group and AML+Ara-C+MSC group were injected intraperitoneally with cytarabine (Ara-C) 60mg/kg on day1-day3 to induce myelosuppression. Mice in the AML+Ara-C+MSC group were injected 3×10 6 UCMSC through the tail vein on day4. We observed the changes in peripheral blood, bone marrow signaling pathways, and AML progression in mice. Results: The experiment found that UCMSC rescued the body weight and peripheral blood. We also found that UCMSC could increase the number of bone marrow CD117 + hematopoietic stem/progenitor cells and CD41 + megakaryocytes through flow cytometry. We verified at protein level that the hematopoiesis-related signaling pathway JAK2/STAT3 was up-regulated in AML+Ara-C+MSC group compared with AML+Ara-C group through flow cytometry and immunohistochemistry. At the same time, the infusion of UCMSC after Ara-C had no influence on disease progression. On day15, the proportion of human CD45 + cells in the bone marrow of between AML+Ara-C+MSC group and AML+Ara-C group was similar, and there was no statistical difference (21.96±3.10 vs. 23.04±1.51; P=0.6792). Conclusion: Infusion of exogenous UCMSC after chemotherapy in PDX models could promote the recovery of hematopoiesis without affecting the efficacy of Ara-C. A reasonable UCMSC infusion scheme is potential to be used in the treatment of AML patients and it requires a lot of preclinical exploration in the future. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Cytarabine (Ara-C) was administrated to mice models to induce myelosuppression.

scholarly journals Comparative in vitro study of the cytotoxicity of gelatine and alginate to human umbilical cord mesenchymal stem cells

2019 ◽  
Vol 52 (1) ◽  
pp. 36
Author(s):  
Nike Hendrijantini
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Colorimetric Assay ◽  
In Vitro Study ◽  
Human Umbilical Cord ◽  
Significant Difference ◽  
Diphenyl Tetrazolium Bromide

Background: Mesenchymal stem cells (MSCs) and scaffold combination constitute a promising approach currently adopted for tissue engineering. Umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are easily obtained and non-invasive. Gelatine and alginate constitute a biocompatible natural polymer scaffold. At present, a cytotoxicity comparison of gelatine and alginate to hUC-MSCs is not widely conducted Purpose: This study aimed to compare the cytotoxicity of gelatine and alginate in hUC-MSCs in vitro. Methods: Isolation and culture were performed on hUC-MSCs derived from healthy full-term neonates. Flow Cytometry CD90, CD105 and CD73 phenotype characterization was performed in passage 4. 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was performed to measure the cytotoxicity. The three sample groups were: (T1) hUC-MSCs with α-MEM (alpha-minimum essential medium) solution as control; (T2) hUC-MSCs with gelatine; (T3) hUC-MSCs with alginate Results: Flow cytometry of hUC-MSCs displayed positive CD90, CD105 and CD73 surface markers. Gelatine and alginate had no effect on the viability of hUC-MSCs and no statistically significant difference (p>0.05) of cytotoxicity between gelatine and alginate to hUC-MSCs. Conclusion: Gelatine and alginate proved to be non-toxic to hUC-MSCs in vitro.

scholarly journals The assessment of CD146-based cell sorting and telomere length analysis for establishing the identity of mesenchymal stem cells in human umbilical cord

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 126 ◽  
Author(s):  
Dimitrios Kouroupis ◽  
Sarah M. Churchman ◽  
Dennis McGonagle ◽  
Elena A. Jones
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Cell Sorting ◽  
Adult Stem Cells ◽  
Human Umbilical Cord ◽  
Length Analysis

Adult stem cells are characterised by longer telomeres compared to mature cells from the same tissue. In this study, candidate CD146+ umbilical cord (UC) mesenchymal stem cells (MSCs) were purified by cell sorting from UC tissue digests and their telomere lengths were measured in comparison to donor-matched CD146-negative fraction.UC tissue fragments were enzymatically treated with collagenase and the cells were used for cell sorting, colony-forming fibroblast (CFU-F) assay or for long-term MSC cultivation. Telomere lengths were measured by qPCR in both culture-expanded MSCs and candidate native UC MSCs. Immunohistochemistry was undertaken to study the topography of CD146+ cells.Culture-expanded UC MSCs had a stable expression of CD73, CD90 and CD105, whereas CD146 declined in later passages which correlated with the shortening of telomeres in the same cultures. In three out of four donors, telomeres in candidate native UC MSCs (CD45-CD235α-CD31-CD146+) were longer compared to donor-matched CD146- population (CD45-CD235α-CD31-CD146-). The frequency of CD45-CD235α-CD31-CD146+ cells measured by flow cytometry was ~1000-fold above that of donor-matched CFU-Fs (means 10.4% and 0.01%, respectively). CD146+ cells were also abundant in situ having a broad topography including high levels of positivity in muscle areas in addition to vessels.

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